Characterization of a novel Negevirus and a novel Bunyavirus isolated from Culex (Culex) declarator mosquitoes in Trinidad.

Pools of mosquitoes were tested for insect-specific viruses using cytopathic effect (CPE) assays on Aedes albopictus (C6/36) cells. Illumina sequencing of RNA from pool TR7094, which produced extensive CPE 2 days post-infection, yielded the complete genome sequences of a previously unknown Bunyavirus, designated Cumuto virus (CUMV), and a second virus designated Wallerfield virus (WALV). WALV shared highest amino acid identity (60.1 %) with Dezidougou virus from Côte d'Ivoire, a positive-sense, single-strand RNA, insect-specific virus belonging to the newly proposed genus Negevirus associated with mosquitoes and phlebotomine sandflies. The S, M and L segments of CUMV were most closely related to those of Gouleako virus, also from Côte d'Ivoire (amino acid identities of 36 %, 38% and 54 % respectively). Neither virus produced CPE on vertebrate cells, or illness in newborn mice. Isolation and characterization of these viruses increase our knowledge of the geographical distribution, diversity and host range of mosquito-specific bunyaviruses and negeviruses.

Molecular epidemiology of group C viruses (Bunyaviridae, Orthobunyavirus) isolated in the Americas.

To date, no molecular studies on group C viruses (Bunyaviridae, Orthobunyavirus) have been published. We determined the complete small RNA (SRNA) segment and partial medium RNA segment nucleotide sequences for 13 group C members. The full-length SRNA sequences ranged from 915 to 926 nucleotides in length, and revealed similar organization in comparison with other orthobunyaviruses. Based on the 705 nucleotides of the N gene, group C members were distributed into three major phylogenetic groups, with the exception of Madrid virus, which was placed outside of these three groups. Analysis of the Caraparu virus strain BeH 5546 revealed that it has an SRNA sequence nearly identical to that of Oriboca virus and is a natural reassortant virus. In addition, analysis of 345 nucleotides of the Gn gene for eight group C viruses and for strain BeH 5546 revealed a different phylogenetic topology, suggesting a reassortment pattern among them. These findings represent the first evidence for natural reassortment among the group C viruses, which include several human pathogens. Furthermore, our genetic data corroborate previous relationships determined using serologic assays (complement fixation, hemagglutination inhibition, and neutralization tests) and suggest that a combination of informative molecular, serological, and ecological data is a helpful tool to understand the molecular epidemiology of arboviruses.

[Concomitant activity of 2 bunyaviruses in horses in Argentina]. / Actividad concomitante de dos Bunyavirus en caballos de la Argentina.

A serologic survey of horses for Kairi (KRI) and Cache Valley (CV), two related Bunyaviruses, was conducted simultaneously in Cordoba and Santa Fe provinces, Argentina, during late 1983 and 1984. The prevalence of neutralizing antibodies only for KRI was 13.3% and only for CV was 40.0%; but if the total positive sera for KRI and CV were taken into account, the prevalence reached 48.3 and 75.0%, respectively. The prevalence for CV was higher than for KRI in Cordoba (p less than 0.01), but both were similar in Santa Fe province. The demonstration of seroconversion in horses of the two zones for both viruses indicates that these viruses have a concomitant activity. The infection rates (number of infections per 100 horses-month) were very high in Cordoba (4.4 and 7.1 for KRI and CV) but also in Santa Fe (2.9 and 9.5 for the two viruses respectively), without significant difference in each province. Despite this high activity, no signs of illness or death imputed to these viruses were registered, in these areas during the period of observation. This apparent absence of associated equine disease may be the consequence of the low or null virus pathogenicity or the underrecognition or underreporting of the clinical cases.

Uso de células de Aedes albopictus C6/36 na propagaçäo e classificaçäo de arbovirus das famílias Togaviridae, Flaviviridae, Bunyaviridae e Rhabdoviridae / The use of Aedes albopictus C6/36 cells in the propagation and classification of arbovirus of the Togaviridae, Flaviviridae, Bunyaviridae and Rhabdoviridae families

Colônias de células de mosquito Aedes albopicus C6/36 foram infectadas com 23 arbovirus, sendo 19 destes existentes no Brasil, pertencentes às famílias Togavitidae, Flaviviridae, Bunyaviridae e Rhabdoviridae. A Replicaçäo viral foi detectada por imunofluorescência indireta com todos os vírus estudados enquanto que o efeito citopático foi observado durante a infecçäo por alguns deste. No teste de imunofluorescência indireta utilizou-se fluidos ascíticos imunes de camundongos, especificos para os vírus estudados. A replicaçäo viral caracterizada por grande produçäo de antígeno recomenda a utilizaçäo de células C6/36 na propagaçäo e em tentativas de isolamento desses arbovírus. A técnica de imunofluorescência ofereceu subsídios na classificaçäo e identificaçäo de vírus que replicam nestas células

[The use of Aedes albopictus C6/36 cells in the propagation and classification of arbovirus of the Togaviridae, Flaviviridae, Bunyaviridae and Rhabdoviridae families]. / Uso de células de Aedes albopictus C6/36 na propagação e classificação de arbovírus das famílias Togaviridae, Flaviviridae, Bunyaviridae e Rhabdoviridae.

C6/36 Aedes albopictus cells were infected with Brazilian arbovirus from the families Togaviridae, Flaviviridae, Bunyaviridae and Rhabdoviridae. Replication was obtained with all the studied viruses and cytopathic effect was observed with some. Viral antigen was assayed in C6/36 cell cultures for antigen was assayed in C6/36 cells by an indirect immunofluorescence test using specific mouse immune ascitic fluid. Antigen production was detected in C6/36 cells infected with all the studied viruses. The author recommends the inoculation of C6/36 cell cultures for isolation of virus from the four studied families. The immunofluorescence technique is an important tool for classification and identification of virus growing in C6/36 cells.

Characterization of five new phleboviruses recently isolated from sand flies in tropical America.

Five new phlebotomus fever virus serotypes (Bunyaviridae: Phlebovirus) are described. These viruses, designated Ambe, Ixcanal, Mariquita, Armero, and Durania, were isolated from sand flies (Diptera: Psychodidae) collected in Brazil, Colombia, and Guatemala. Two of the agents were recovered from pools of male sand flies. The new viruses are antigenically related to other members of the phlebotomus fever serogroup by immunofluorescence, but are distinct from the other 39 members of this serogroup by plaque reduction neutralization test.

Transovarial transmission of Gamboa virus in a tropical mosquito, Aedeomyia squamipennis.

We report transovarial transmission of Gamboa virus (Bunyavirus) in Aedeomyia squamipennis, a tropical mosquito which is active and bloodfeeding throughout the year. Gamboa virus was isolated during each of the 28 months of the study from every mosquito stage, including eggs, demonstrating that vertical transmission is a maintenance mechanism of this virus. The overall minimum infection rate was 5.1/1,000 mosquitoes. Identification of the 567 isolates by neutralization indicated that greater than or equal to 2 serotypes or subtypes of Gamboa virus circulate at the study site.

Characterization of eight new phlebotomus fever serogroup arboviruses (Bunyaviridae: Phlebovirus) from the Amazon region of Brazil.

Eight new members of the phlebotomus fever arbovirus serogroup (family Bunyaviridae; genus Phlebovirus) from the Amazon region of Brazil are described. One serotype was recovered from a febrile patient, three from small wild animals and four from sand flies. A small serum survey carried out with the human isolate, Alenquer virus, suggests that it rarely infects man. Complement-fixation and plaque reduction neutralization tests were done, comparing the eight new viruses with other members of the phlebotomus fever serogroup. A close antigenic relationship was demonstrated between one of the new agents (Belterra) and Rift Valley fever virus. This finding is of considerable interest and deserves further investigation. Addition of these eight new viruses to the genus Phlebovirus brings to 14 the number of serotypes known to occur in the Amazon region and to 36 the total number reported worldwide. More detailed clinical and epidemiological studies should be conducted in Amazonia in order to define the public health impact caused by phleboviruses.

Viruses isolated from Aedeomyia squamipennis mosquitoes collected in Panama, Ecuador, and Argentina: establishment of the Gamboa serogroup.

Twenty-four virus strains were isolated from Aedeomyia squamipennis mosquitoes collected in Ecuador. One additional strain each was isolated from this species from Panama and ARgentina. All 26 isolates were shown to be related serologically to prototype Gamboa virus, originally isolated from Ad. squamipennis mosquitoes collected in Panama. Antigenic comparisons of eight strains, including prototype Gamboa virus, indicated the existence of four distinct viruses. Neutralization tests with sera from a variety of mammalian and avian species from Argentina provided further evidence that Gamboa serogroup viruses are transmitted between Ad. squamipennis and birds.